Xenotransplantation of the endocrine pancreas

C H A P T E R

31 Xenotransplantation of the endocrine pancreas Benjamin Smood⁎, Rita Bottino†,‡,§,¶, David K.C. Cooper|| ⁎

University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA, †Institute of Cellular Therapeutics, Pittsburgh, PA, USA, ‡Allegheny-Singer Research Institute, Pittsburgh, PA, USA, §Allegheny Health Network, Pittsburgh, PA, USA, ¶Carnegie Mellon University, Pittsburgh, PA, USA, ||Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA O U T L I N E Introduction A novel approach to discrepancies in supply and demand Defining success

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A brief history of xenotransplantation Earliest attempts in xenotransplantation and xenotransfusion Origins of endocrine transplantation Advancing to the modern era of xenotransplantation

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Optimizing the pig-to-NHP model The pig-to-NHP as the preferred preclinical model

426 426

423 424

424 424 425

Laying the foundation: preclinical studies in islet xenotransplantation 427 Hurdles to free and encapsulated islet xenotransplantation 427 Encapsulation 427 Overcoming immediate host responses: pharmacotherapies to prevent the instant blood-mediated inflammatory reaction 428 Ideal placement of free islets in xenotransplantation 428 Composite islet-kidney grafts 430 Cotransplantation of islet xenografts and “regulatory” cells 430

Introduction A novel approach to discrepancies in supply and demand In the modern era of diabetic care, life expectancy for men and women with diabetes reaching 20  years of age is 11.1 and 12.9  years shorter, respectively, than

Transplantation, Bioengineering, and Regeneration of the Endocrine Pancreas, Volume 2 https://doi.org/10.1016/B978-0-12-814831-0.00031-2

Genetic modifications to combat IBMIR Control of the T cell response Will sensitization to human leukocyte antigens be detrimental to islet xenotransplantation? Will sensitization to pig antigens preclude subsequent islet allotransplantation? The induction of immune tolerance: The “Holy Grail” of transplantation Improving function of porcine islets

432 434 435 435 435 436

Justification for translation to clinical trials Lessons from early clinical trials Establishing safety Determining efficacy Patient selection

436 436 437 438 438

Future directions Research priorities Conclusions

438 438 439

Acknowledgment

439

Conflict of interest

439

References

439

the general population.1,2 Clinical advances appear to have reached a plateau in reducing morbidity and mortality in diabetic patients, and innovation is required to advance this field and improve the quality of life.3 Pancreatic transplantation remains the gold standard of treatment as a cure for type 1 diabetes (T1D) since its first application in 1966.4 However, the ubiquitous shortage of pancreas donors and a decrease in the number of

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© 2020 Elsevier Inc. All rights reserved.

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31.  Xenotransplantation of the endocrine pancreas

whole pancreas transplants in the past decade has called for exploration of other approaches to β-cell replacement therapy. Among these are free islet allotransplantation, encapsulation technologies, β-cell regeneration, stem cell-derived β-cells, the artificial pancreas, threedimensional (3D)-printed organs, and islet xenotransplantation. Each of these topics is covered extensively in this book, and here we focus on islet xenotransplantation, specifically on the experience in the pig-to-­nonhuman primate (NHP) model and its potential translation into clinical practice. Opinion leaders of the International Pancreas and Islet Transplant Association and The Transplantation Society (IPITA-TTS) anticipate that, within 5–10 years, islet allotransplantation will be the preferred therapeutic procedure for β-cell replacement due to its improved metabolic efficacy and superior safety profile over conventional pancreas transplantation.2 Results from a multicenter phase III clinical trial indicate that islet allotransplantation may indeed prove to become the gold standard of care for patients with severe hypoglycemia unawareness.2,5,6 However, as is the case with all organ allotransplantation, the shortage of organs from deceased donors limits the number of transplants that can be carried out each year. Moreover, islet allotransplantation may require the transplantation of islets from two or more donors to achieve normoglycemia.5,7 The number of pancreases available for islet allotransplantation in diabetic patients, therefore, will never be sufficient to treat all but a small percentage of those who might benefit. As an alternative solution, the safety and efficacy of xenogeneic transplantation have been studied extensively in the past several years in NHPs.6,8–25 Despite a decade of experience, obstacles remain to be overcome before this novel therapeutic approach can become standard of care.

Defining success Essential to translating porcine islet xenotransplantation into clinical practice is a clear definition of success. Proposed end points have ranged from the detection of increased levels of C-peptide or reduced hemoglobin A1c, to ambitions of freedom from episodes of both hyperglycemia and hypoglycemia, reduced secondary complications, and insulin independence. Currently, the IPITA-TTS Executive Summary recommends that the primary goal of islet allotransplantation should be optimal glycemic control without severe hypoglycemia, rather than insulin independence.6 A specific goal for xenotransplantation has not been defined, but it is prudent to consider it to be the same as for allotransplantation. In order for xenotransplantation to be a practical and sustainable solution to patients affected by T1D with

high risk for hypoglycemia, its implementation must be cost-effective and safe. The ensuing sections outline the historical advances and accelerating progress in this area of study, including a review of clinical trials and future directions in research and regulation.

A brief history of xenotransplantation Earliest attempts in xenotransplantation and xenotransfusion A historical perspective outlining the evolution of xenotransplantation is helpful to provide context for the current state of islet xenotransplantation. Transplantation of tissues or organs has been proposed and/or attempted for centuries. Reports from India dating to c.600 BC illustrate physicians using skin autografts to repair amputated noses.26 Circa 225 BC, a Chinese surgeon was reported to successfully exchange the hearts of soldiers in hopes to restore their Yin and Yang.26,27 Similar legends are pervasive throughout human history. Albeit unlikely successful outside of folklore and mythology in its earliest attempts, it exemplifies the rich and ancient history of human fascination with organ replacement as a medical cure.26,28 Scientifically documented xenotransplantation in humans was born in 1667 when Jean Baptiste Denis and Paul Emmerez transfused the blood of a lamb into a 15-year-old feverish boy, and later that year transfused calf blood into a mentally ill man in an attempt to cure him.26,29,30 With today’s understanding of cross-species immune reactivity, it is not surprising that their results were mixed at best, ultimately leading to years of French and English parliaments prohibiting xenotransfusions.26,28 Nevertheless, their brazenness marked the earliest attempts in the field that would eventually become known as xenotransplantation.

Origins of endocrine transplantation A historical timeline of pioneering work in xenotransplantation, including skin, cornea, kidney, liver, and heart transplantation, is beyond the scope of this chapter. However, the work of Charles-Edouard Brown-Séquard and Serge Voronoff using testicular xenografts for “rejuvenation therapy” is, perhaps surprisingly, of particular relevance to islet transplantation.26,28,31,32 The FrenchAmerican physician and physiologist, Brown-Séquard, was among the earliest to hypothesize that substances secreted into the blood, which we now recognize as ­hormones, could affect distant organs. Among his other contributions to establishing the field of endocrinology, in 1889 at the age of 72 he injected himself with an extract of crushed guinea pig and canine testicles which he

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A brief history of xenotransplantation

self-reported (at the Societie de Biologie meeting in Paris) to have improved his physical and sexual prowess.31,32 Similar experiments gained popularity between 1920 and 1950 as Serge Voronoff, a Russian émigré working in Paris, transplanted pieces of chimpanzee or baboon testicles into human testicles as another form of “rejuvenation therapy.”28 Hundreds, if not thousands, of these operations were performed during the early 20th century on both sides of the Atlantic, and with surprisingly uncommon reports of serious complications. By the end of his life, Voronoff and his techniques were largely discredited and ridiculed by the scientific community.26 Despite the fact that any benefit was likely only secondary to psychological and placebo effects, it would be remiss not to acknowledge that Voronoff’s procedures were visionary and ahead of his time. Furthermore, even in these earliest days of cross-species transplantation, he was concerned that the supply of primates could limit large-scale application.26,28 Widely considered a medical charlatan, controversial surgeon John Brinkley, chose the goat as the “donor” of testicles, but was eventually driven out of business by the American Medical Association.33 The concept of xenotransplantation has evolved for 350 years, and now in the modern era of endocrine xenotransplantation we are becoming successful in the very principles that Brown-Séquard and Voronoff envisioned; there is legitimate feasibility for ameliorating hormonal deficiencies by transplanting pancreatic islets to individuals with severe T1D.28 Early attempts to treat diabetes by transplanting sheep or feline pancreatic fragments were reported in the medical literature in 1883 and 1903.34,35 None of the attempts succeeded, resulting in the deaths of the patients with no improved metabolic function. The earliest studies in xenotransplantation focused on cells and tissues, rather than whole organ transplants, due to shortcomings in revascularization techniques and bleeding control. Still, even after successfully overcoming these anatomic and procedural hurdles, physiologic and immunologic obstacles remained.

Advancing to the modern era of xenotransplantation Early experiments into whole organ xenotransplantation were performed in the late 19th and early 20th centuries, and were focused largely on kidney xenotransplantation due to the simplicity of the single arterial supply, the unavailability of human organs, and the fact that dialysis was not yet in use. Advances in vascular anastomosis ushered in a new, albeit short, era of xenotransplantation. Repeated failures often cited thrombosis and fatal complications.26,36–38 Mathieu Jaboulay, who perfected an anastomosis technique in 1906 for heterotopic kidney xenotransplantation, hypothesized that xenografts, unlike autografts, likely created conditions that

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promoted coagulation, but he failed to propose a distinct mechanism.26,36 These repeated hurdles to success culminated with Harold Neuhof’s attempt to transplant a lamb kidney to a man with mercury poising in 1923.26,38 The patient died 9 days after the procedure. It would be 40 years until xenotransplantation was attempted again in 1964.26 Keith Reemtsma transplanted a pair of kidneys from a chimpanzee to a human in 1964, marking the first use of immunosuppressive therapy in such a procedure. He went on to carry out a total of six chimpanzee kidney transplants in his patients, one of whom lived for a remarkable 9  months before dying relatively suddenly from what was believed to be a septic complication.26,39–41 Encouraged by the success of Thomas Starzl with the world’s first liver allotransplant program, a British surgeon, Roy Calne, established the world’s second, and first such program in Europe. To this point, clinical trials were largely undertaken without previous experience in other species. However, in 1968 the paradigm for xenotransplantation shifted to consider trials in NHPs before attempting procedures in humans. Calne reported his experience with pig liver transplantation in baboons, rhesus monkeys, and a chimpanzee, in part establishing the pig-to-NHP model that has since become standard practice.42,43 Continued efforts to control the immune response to allografts led to the discovery of cyclosporine by JeanFrancois Borel in 1976.44 With renewed hope for the success of xenotransplantation in this “cyclosporine era,” alongside promise in preclinical studies, exploration of the field became more ambitious. By 1984, Leonard Bailey had demonstrated an average survival of 72 days of newborn lamb-to-goat cardiac xenotransplants.45 This resulted in the famous xenotransplantation of a baboon heart into a 12-day-old human infant with hypoplastic left heart syndrome. The child, “Baby Faye,” died 20 days after the operation, which, alongside a number of other failures in xenotransplantation models, temporarily halted further attempts at xenotransplantation despite improvements in immunosuppressive therapy. With the introduction of the new immunosuppressant, tacrolimus (FK506), in 1992 Thomas Starzl reported the 70-day survival of a man who received a baboon liver, which reinvigorated academic interest in xenotransplantation.46,47 Despite this progress, it was evident that immunological and physiological barriers remained to be overcome to move the field forward. Progress in islet xenotransplantation has benefited from advances in islet biology, and from the development of methods of islet isolation for islet allotransplantation. Technical approaches to isolate intact islets from the pancreas of rodents were developed in the mid-1960s and the introduction of density gradients to separate islets from exocrine cells occurred soon after.48–50 However, it took an additional 20 years before Ricordi introduced the

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automated method for the isolation of islets from human pancreases.51 This watershed event allowed multiple centers to initiate programs of islet allotransplantation in patients with T1D. It also paved the way to optimize methods for the isolation of porcine islets.52 Citing the established isolation methods for largescale fetal porcine islet-like cell clusters, between 1990 and 1993, Swedish surgeon, Carl-Gustav Groth, and his colleagues performed the first clinical pig islet xenotransplants.52,53 Fetal porcine islet-like cell clusters were placed intraportally or underneath the kidney capsule (in patients receiving renal allografts). The immunosuppressive regimen consisted of cyclosporine, prednisolone, and azathioprine. Publishing their results in 1994, this work demonstrated that at least some porcine islets could survive in humans, despite a lack of clear clinical benefit. Groth’s work served as a harbinger for the preclinical and clinical studies that have been carried out since that time, which have sought to combat graft rejection and improve endocrine function following islet xenotransplantation. These studies offer hope of improving the quality of life and reducing morbidity and mortality in patients with T1D, particularly in those with severe hypoglycemic unawareness. The pig-to-NHP setting has provided the most promising model to translate this therapy to humans.

TABLE 1  Advantages and disadvantages of the pig as a potential source of organs and cells for humans, in contrast to the baboon in this role Pig

Baboon

Availability

Unlimited

Limited

Breeding potential

Good

Poor

Period to reproductive maturity

4–8 months

3–5 years

Length of pregnancy

114 ± 2 days

173–193 days

Number of offspring

5–12

1–2

Growth

Rapid (adult human Slow (9 years to reach max. size) size by 6 months)a

Size of adult organs

Adequate

Inadequateb

Cost of maintenance

Significantly lower

High

Anatomical similarity to humans

Moderately close

Close

Physiological similarity to humans

Moderately close

Close

Immune system relation to humans

Distant

Close

Knowledge of tissue typing Considerable (in selected herds)

Limited

Necessity for blood type compatibility with humans

Probably unimportant

Important

Experience with genetic engineering

Considerable

None

The pig-to-NHP as the preferred preclinical model

Risk of transfer of infection (xenozoonosis)

Low

High

The pig-to-NHP model allows study of both efficacy and safety of islet xenotransplantation.6,8,11–13,15–17,19–22,54 NHPs are the optimal preclinical hosts in studying xenotransplantation because their immune system mimics that of humans.55 While NHPs may have differences in their response to biological or pharmacologic treatments and glucose metabolism when compared to humans, both monkeys and baboons are considered appropriate for use in these studies.54,56 However, one drawback is that NHPs do not develop autoimmune T1D.54,57,58 As such, a state of diabetes has to be (and has successfully been) induced using streptozotocin to provide a relevant model of islet xenotransplantation.59–61 The pig is regarded as the most likely potential islet donor for several compelling reasons (Table 1). Porcine insulin was used for nearly a century to treat diabetes successfully before the introduction of recombinant human insulin, and it differs by only a single amino acid from human insulin.62–64 The benefits of porcine donors compared to other species, such as NHPs, that are genetically closer to humans are several, and include a relatively low cost of maintenance, which makes large-scale application of unlimited islet cells feasible (Table 1).55,65

Availability of specific pathogen-free animals

Yes

No

Public opinion

More in favor

Mixed

Optimizing the pig-to-NHP model

a

Breeds of miniature swine are approximately 50% or less of the weight of domestic pigs at birth and sexual maturity, and reach a maximum weight of approximately 30% or less of standard breeds. b The size of certain organs, e.g., the heart, would be inadequate for transplantation into adult humans.

Research groups have attempted to identify the strain of pig that produces the highest quality and quantity of islets, but consensus has yet to be reached regarding the ideal demographics for the pig donor.66–71 Concerning the optimum age of the pig, there are advantages and disadvantages to both fetal/neonatal and adult pig islets (Table 2).72 Adult pigs provide more islets and these are more mature, and thus secrete insulin immediately (Fig.  1). They express less of the immunogenic epitope galactose-α1,3-galactose (Gal) compared to neonatal islets, but this can be negated by the use of islets from α1,3-­galactosyltransferase gene-knockout (GTKO) pigs.55 Neonatal islets may

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Laying the foundation: preclinical studies in islet xenotransplantation

TABLE 2  Advantages and disadvantages of embryonic, fetal, neonatal, and adult pig islets for clinical xenotransplantationa Embryonic

Fetal

Neonatal

Adult

Isolation procedure

Simple

Simple

Simple

Difficult

Cost of islet isolation

Low

Low

Low

High

Gal expression

High

High

High

Low

Islet yield/pancreas (IEQs)

N/A

~ 8000

25,000–50,000

200,000–500,000

Beta cells (% of islet cells)

N/A

10%

25%

> 70%

Insulin production

Delayed > 3 months

Delayed > 2 months

Delayed > 1 month

Immediate

Proliferation in vivo

Yes

Yes

Yes

Little/none

Tumorigenicity

Possible

Low

Low

None

Risk of pathogen transmission

Low

Low

Low

Low

Cost of housing pig until islets utilized

Low

Low

Low

High

Gal = galactose-α1,3-galactose; N/A = not available. a Modified from Dufrane D, Gianello P. Pig islet for xenotransplantation in human: structural and physiological compatibility for human clinical application. Transplant Rev (Orlando) 2012;26(3):183–188; Nagaraju S, Bottino R, Wijkstrom M, Trucco M, Cooper DK. Islet xenotransplantation: what is the optimal age of the islet-source pig? Xenotransplantation 2015;22(1):7–19; Park CG, Bottino R, Hawthorne WJ. Current status of islet xenotransplantation. Int J Surg 2015;23(Pt B):261–266; Zhu HT, Yu L, Lyu Y, Wang B. Optimal pig donor selection in islet xenotransplantation: current status and future perspectives. J Zhejiang Univ Sci B 2014;15(8):681–691.

be less susceptible to anoxic injury posttransplant73. All things considered, neonatal islet transplants have had promising results, and are currently considered the preferred age as sources of islets for clinical application.6,8,11,14,15,55,74–76 Of course, fewer islets can be obtained from young pigs, which may require a greater number of donor pigs. The islets also require a latent period to mature, but they

proliferate after transplantation. It is estimated that neonatal pigs can provide 25,000–30,000 islets per piglet. Patients might require roughly 10,000–20,000 islet equivalents (IE)/kg for effective treatment; thus a 70-kg patient would need approximately 25–50 piglet donors (10,000 IE/kg × 70 kg). With each litter consisting of perhaps 4–10 piglets, this is certainly feasible.77 Given that islet xenografts target a serious and chronic clinical condition, the number of donors required to treat “brittle diabetes” is not an unreasonable consideration and may well prove cost-effective.78

Laying the foundation: preclinical studies in islet xenotransplantation Hurdles to free and encapsulated islet xenotransplantation A major challenge of porcine islet transplantation was the early inflammatory/immune response to the graft.6,79 Can modern medicine adequately control this response in cross-species transplantation? Advances in genetic modification of donors and in immunosuppressive therapy to the host suggest the answer is “yes.”

Encapsulation

FIG. 1  Adult pig islets after isolation. Adult pig islets stained in red with dithizone after isolation and purification (Magnification 40 ×).

Some have advocated for the use of “encapsulated” islet cells in xenotransplantation—a method to isolate donor and host cells from one another. This topic in relation to allotransplantation is discussed elsewhere in this

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book, and so only brief mention of it will be included here. Modern biomedical engineering is currently investigating micro- and macrostructures that could protect against host immune responses while maintaining oxygenation and nutrition of the encapsulated islets, with some partial success (Table 3).21,22,25,80–84 Among the chief arguments in favor of this method is that it could theoretically eliminate or at least reduce the need for host immunosuppressive therapy by preventing the interface of cross-species tissues and blood. However, materials that allow the secretion of insulin will likely allow some cytokines and chemokines to leak across the membrane with exposure to islets, causing destructive immunologic reactions. Recent studies have suggested that some exogenous immunosuppressive therapy may be necessary, negating the full benefit of encapsulation.23,55,77,80,85–89

Overcoming immediate host responses: pharmacotherapies to prevent the instant blood-mediated inflammatory reaction If pig islets are transplanted into the portal vein (which is the current site of choice for allotransplantation), the instant blood-mediated inflammatory reaction (IBMIR) poses a significant challenge to free islet xenotransplantation.6,61,79,90,91 Contact between blood and islets (particularly xenogeneic islets) produces an inflammatory reaction that activates the complement and coagulation systems, leading to rapid destruction of the islets. Among the complex pathophysiological events that take place during IBMIR, upregulation of the expression of tissue factor on the islets plays a significant initiating role (Fig.  2).55,79,80,90–94 There are extensive in  vivo and in  vitro data confirming the activation of

complement and coagulation in response to the “foreign” islets.20,95,96 Evidence now suggests that IBMIR may have much in common with hyperacute rejection in whole organ xenotransplantation, and is at least in part secondary to the binding of the host’s natural anti-pig antibodies to the islets (Fig.  3), initiating activation of the complement and coagulation cascades.80,95,97,98 Thus, targeting the roles of antibody, complement, and coagulation is paramount in making free islet xenotransplantation a viable therapy. Pharmacologic strategies to reduce IBMIR have targeted complement activation with cobra venom factor (CVF), various complement inhibitors, and ­anti-­inflammatory agents, such as monoclonal antibodies directed to tumor necrosis factor-α (TNF-α).79,93,98–103 The immediate coagulation that occurs in IBMIR has been combatted (relatively unsuccessfully) with heparin or thrombin inhibitors, anti-platelet agents, and by directly targeting tissue factor with anti-tissue factor antibodies.55,91,99,104–109 Pretreatment of islet xenografts with nicotinamide can reduce tissue factor expression, and partially protect against IBMIR.110,111 However, each of these methods raises clinical concerns, including the detrimental effects of complement suppression and potential bleeding complications.

Ideal placement of free islets in xenotransplantation The portal vein/liver is currently the preferred clinical site in free islet xenotransplantation, with most success in clinical practice.7,112–115 However, the liver is less than an ideal location.113 After transplantation, there is a potential risk of hemorrhage and portal vein thrombosis.

TABLE 3  Experience with the xenotransplantation of encapsulated pig islets in non-immunosuppressed NHPs Reference

Donor/recipient

Device

Implant site

Maximal graft survival

Sun et al.25

Adult/diabetic CM

Alginate-PLL-alginate

Peritoneal cavity

120–803 days

7/9 recipients achieved normoglycemia

Elliott et al.82

NICCs/nondiabetic CM Alginate-PLO-alginate

Peritoneal cavity

> 8 weeks

Insulin-positive islets in retrieved capsules

Elliott et al.83

Clinical outcome

NICCs/diabetic CM

Alginate-PLO-alginate

Peritoneal cavity

> 36 weeks

Reduced insulin requirement by 16%

84

Adult/nondiabetic CM

High-M alginate

Kidney capsule

> 180 days

Urine porcine C-peptide-positive

21

Dufrane et al.

Adult/diabetic CM

Alginate MCD

Subcutaneous

> 6 months

Diabetes correction > 6 months

22

Adult + pig MSCs/ diabetic CM

Alginate MCD

Subcutaneous

> 32 weeks

Diabetes correction > 32 weeks

Dufrane et al.

Veriter et al.

CM, cynomolgus monkey; MCD, monolayer cellular device; MSC, mesenchymal stromal cells; NICCs, neonatal islet-like clusters; PLL, poly-l-lysine; PLO, poly-l-ornithine. Modified from Zhu HT, Lu L, Liu XY, et al. Treatment of diabetes with encapsulated pig islets: an update on current developments. J Zhejiang Univ Sci B. 2015;16(5):329-343.

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Laying the foundation: preclinical studies in islet xenotransplantation

Islet infusion into the portal vein

IBMIR

Islet Thrombus formation (Thrombin)

Lysis (C3a, C5a, MAC...)

Platelet Fibrinogen Leukocytes TF FVIIa

Coagulation activation & platelet activation and aggregation

Complement activation & infiltration of leukocytes

iC3b Red blood cell

FIG. 2  Overview of IBMIR. The contact between blood and islets triggers the activation of coagulation that is mediated through tissue factor (TF). As a result, thrombin is generated, leading to fibrinogen deposition. Attachment of platelets to islets further increases the procoagulant effect. Complement (iC3b) is deposited on the islet surface, C3a and C5a are activated, attract leukocytes, and promote formation of membrane attack complex (MAC) which mediates the lysis of islets. (FVIIa = activated coagulation factor VII). Reproduced with permission from Liu Z, Hu W, He T, Dai Y, Hara H, Bottino R, Cooper DKC, Cai Z, Mou L. Pig-to-primate islet xenotransplantation: past, present, and future. Cell Transplant. 2017;26(6):925–947.

Oxygen tension in the portal vein is low, which may trigger islet apoptosis. Direct exposure of islets to the blood results in a substantial loss of islets from IBMIR. It is estimated that IBMIR reduces the number of successfully transplanted islets in the portal system by 60% within the first few hours or days.6,93,98,116–119 Furthermore, the islet graft cannot be retrieved, and liver biopsies usually do not yield sufficient islets for analysis. While better control of IBMIR and graft rejection could improve outcomes of portal transplantation (described above), alternative sites for free islet transplantation must continue to be explored to improve the clinical outcome.6,55,113 Although the pancreas may be considered the natural site for islet transplants, it is not easily accessible, and the risk of pancreatitis would be significant. Therefore,

alternative sites (other than the portal vein) have been explored (Table 4).55,113 These studies have been directed to sites where the islets are not immediately exposed to blood, and thus protected from IBMIR/early graft loss. Potential transplant sites include the gastrointestinal submucosal space, omental pouch, striated muscle, and bone marrow.55,120–127 Although a current trial is examining the omentum as an alternative site for islet allotransplantation, the other locations have been largely studied in small mammals or pigs.128,129 Islet transplants into the renal subcapsular space in animals under optimized protocols have demonstrated some success as an alternative to intraportal islet infusion, but have shown limited success in humans.130–139 Failures are likely due to islet ischemia after transplantation.55,130,131

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FIG. 3  Binding of human IgM and IgG antibody to pig islets (xenogeneic) (A–B) and to human islets (allogeneic) (C–D). IgM (green, A, C), IgG (green, B, D), insulin (red), nucleus (DAPI/blue). Yellow indicates colocalization of insulin and IgM/IgG. The greatly increased binding of human IgM and IgG to pig islets (compared to human islets) is obvious. Reproduced with permission from van der Windt DJ, Marigliano M, He J, Votyakova TV, Echeverri GJ, Ekser B, Ayares D, Lakkis FG, Cooper DK, Trucco M, Bottino R. Early islet damage after direct exposure of pig islets to blood: has humoral immunity been underestimated? Cell Transplant 2012;21(8):1791–1802.

Composite islet-kidney grafts One mechanism to increase the speed of revascularization and engraftment lies in composite islet-kidney transplants. Pig islets have been transplanted under the kidney capsule in autologous or syngeneic settings of pig littermates, allowing revascularization and proliferation of islets in the absence of immune and inflammatory responses.132–135 Some weeks later, the composite islet-kidney graft was transplanted into the ultimate pig recipient which, if MHC mismatched with the composite graft donor tissue, received immunosuppressive therapy. Successful engraftment and immediate function of both transplanted tissue/organ proved the validity and clinical potential of the approach. There has also been success in the immunosuppressed NHP model.136,137 A xenogeneic model would be necessary to make these studies truly clinically relevant. However, a xenogeneic model was originally troubling because of the difficulty in maintaining viability and function of a porcine kidney graft in an NHP. With improved genetic engineering of the organ-source pig (and effective immunosuppressive therapy of the NHP host), this hurdle has now been overcome.140,141 The ultimate goal would

be for the composite islet-kidney to be implanted in patients with end-stage renal disease and diabetes, with the aim of curing the renal failure and controlling glucose metabolism.

Cotransplantation of islet xenografts and “regulatory” cells Cotransplantation has also been studied by combining porcine islets with mesenchymal stem cells (MSCs) or Sertoli cells (SCs). MSCs and SCs have been shown to function across species lines, and possess anti-­ inflammatory, regenerative, and immunomodulatory properties that promote revascularization of islets after xenotransplantation.22,142–150 SCs help create tight junctions to isolate germ cells from the blood in the testis, but there is inconclusive evidence suggesting that SC cotransplantation may improve islet survival in humans.55,85,151–153 Nevertheless, SCs, and especially MSCs, have considerable potential in islet xenotransplantation to diminish acute (and probably chronic) islet graft loss.55,154 The ability to obtain large quantities of porcine MSCs and SCs from the identical, genetically engineered pig islet donor may be a significant advantage.55,142,143

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Gastric submucosal space Pancreas

Liver

Renal capsule Spleen

Skin

Omentum

Muscle

Efficacy of clinical trial

Good

Poor

Not reported

Poor

Limited Experience

Limited experience Not reported

Limited experience

Patient safety

Safe

Safe

Safe

Safe

Safe

Safe

Possible pancreatitis

Safe

Oxygen tension

Low

Not reported

High

Low

Not reported

High

Not reported

Not reported

Vasculature

Rich

Poor

Not reported, but probably rich

Poor

Rich

Rich

Not reported

Rich

Site of insulin released by the graft

Liver

Not reported

Portal vein

Systemic circulation

Portal vein

Portal vein

Not reported

Systemic circulation

Surgery

Invasive, some complications

Invasive

Invasive

Non-invasive

Easy

Easy (endoscopy)

Difficult

Easy

IBMIR

Yes

Not reported

Yes

Not reported

Not reported

Not reported

Not reported

Not reported

Laying the foundation: preclinical studies in islet xenotransplantation

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TABLE 4  Comparison of different sites for free islet xenotransplantation

Modified from van der Windt DJ, Echeverri GJ, Ijzermans JN, et al. The choice of anatomical site for islet transplantation. Cell Transplant 2008;17(9):1005–1014.

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Genetic modifications to combat IBMIR Genetic knockin, knockout, and knockdown models in the pig have been developed to combat IBMIR/ hyperacute rejection.10,20,65,73,155–160 Varying degrees of success have been demonstrated in a number of genetically modified pig strains to prevent immediate islet destruction in the pig-to-NHP model and reduce the need for pharmacologic immunosuppressive therapy to the host.10,15,20,24,55,66,161–163 The identification of Gal on pig cells that is bound by human anti-pig antibodies was a major milestone in combating hyperacute rejection of solid organ xenografts.65,164–168 In 2003, the first GTKO pigs were introduced, lacking the enzyme responsible for adding Gal to the oligosaccharides of pig endothelium and islets that cross-react with host antibodies.167 Since then, two other carbohydrate epitopes have also been targeted, namely Nglycolylneuraminic acid (Neu5Gc) and Sda (the product of β-1,4-N-acetylgalactosaminyltransferase).55,155,156,158,167,169 Since the first successful use of GTKO pigs in islet xenotransplantation, improved methods of genetic manipulation, including the CRISPR technology (Table  5), have facilitated attempts to reduce injury by protecting the graft from the primate immune response (Table 6).61,65,112,167,168,170–180 A recent model developed by Kirk and colleagues demonstrated that islets transplanted from GTKO and WT donor pigs into NHPs developed relatively similar IBMIR responses as measured by insulin, complement, antibodies, neutrophils, and macrophages.181 This model quantitatively highlights the specific difficulties in islet xenotransplantation compared to allotransplantation, demonstrating increased IgM, cellular infiltration, and apoptosis even in GTKO xenogeneic islet donors.182 As an alternative or additional approach, human complement-regulatory proteins, for example, CD46, CD55, and CD59, have been expressed on pig islets TABLE 5  Timeline for application of evolving techniques for genetic engineering of pigs employed in xenotransplantation

TABLE 6  Selected genetically modified pigs currently available for xenotransplantation research Complement regulation by human complement-regulatory gene expression CD46 (membrane cofactor protein) CD55 (decay-accelerating factor) CD59 (protectin or membrane inhibitor of reactive lysis) Antigen deletion α1,3-Galactosyltransferase gene-knockout (GTKO) Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene-knockout (CMAH-KO or Neu5Gc-KO) β4GalNT2 (β-1,4-N-acetylgalactosaminyltransferase) gene-knockout (β4GalNT2-KO) Suppression of cellular immune response by gene expression or downregulation CIITA-DN (MHC class II transactivator knockdown, resulting in swine leukocyte antigen class II knockdown) Class I MHC-knockout (MHC-IKO) HLA-E/human β2-microglobulin (inhibits human natural killer cell cytotoxicity) Human FAS ligand (CD95L) Human GnT-III (N-acetylglucosaminyltransferase III) gene Porcine CTLA4-Ig (cytotoxic T-lymphocyte antigen 4 or CD152) Human TRAIL (tumor necrosis factor-alpha-related apoptosisinducing ligand) Anticoagulation and anti-inflammatory gene expression or deletion von Willebrand factor (vWF)-deficient (natural mutant) Human tissue factor pathway inhibitor (TFPI) Human thrombomodulin Human endothelial protein C receptor (EPCR) Human CD39 (ectonucleoside triphosphate diphosphohydrolase-1) Anti-inflammatory, anti-apoptotic (and anticoagulant) gene expression Human A20 (tumor necrosis factor-alpha-induced protein 3)

Year

Technique

Human heme oxygenase-1 (HO-1)

1992

Microinjection of randomly integrating transgenes

2000

Somatic cell nuclear transfer (SCNT)

Human CD47 (species-specific interaction with SIRP-α inhibits phagocytosis)

2002

Homologous recombination

2011

Zinc finger nucleases (ZFNs)

2013

Transcription activator-like effector nucleases (TALENs)

2014

CRISPR/Cas9a

a

Porcine asialoglycoprotein receptor 1 gene-knockout (ASGR1-KO) (decreases platelet phagocytosis) Human signal regulatory protein α (SIRPα) (decreases platelet phagocytosis by ‘self’ recognition) Prevention of porcine endogenous retrovirus (PERV) activation PERV siRNA

CRISPR/Cas9, clustered randomly interspaced short palindromic repeats and the associated protein 9

PERV KO

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(and organs) and have been demonstrated to protect against the effects of human complement activation.12,55,161,163,183–189 Similarly, transgenic expression of human coagulation-regulatory proteins (e.g., tissue factor pathway inhibitor, thrombomodulin) have reduced the development of thrombotic microangiopathy and consumptive coagulopathy in pig organ grafts in NHP recipients, and may play a role in protecting pig islets from IBMIR.20,55,190–192 A promising milestone for islet xenotransplantation was achieved by van der Windt et al. in 2009, who used islets from pigs expressing hCD46, successfully achieving insulin independence for > 1 year in diabetic monkeys (Fig. 4).12

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Neonatal islets expressing human complement-­ regulatory proteins, CD55 and CD59, on a GTKO background have been shown to attenuate IBMIR-associated pathological events in immunosuppressed baboons, with reduced complement activation and thrombin generation.193 Using multiple human transgenes, including complement and coagulation inhibitors, Bottino et  al. further demonstrated modulation of IBMIRmediated early islet loss, even though this did not consistently translate into better long-term outcomes.20 Simultaneous transgenic modifications in islet donors for GTKO/hCD46/CMAH (Neu5Gc)-KO demonstrated a near-complete reduction in IgM and IgG responses.

FIG.  4  (A) Blood glucose and pig C-peptide levels in a streptozotocin-induced diabetic cynomolgus monkey before and after intraportal transplantation of islets from a pig expressing the human complement-regulatory protein, CD46. No exogenous insulin was administered after the transplant. The normoglycemic monkey was electively euthanized after 12 months. Day 0 = day of islet transplantation. (B) Insulin immunostaining (in red) of a liver section in a monkey recipient of islets from a pig transgenic for human CD46, showing a healthy pig islet 12 months after transplantation. (Magnification × 200). Reproduced with permission from van der Windt DJ, Bottino R, Casu A, Campanile N, Smetanka C, He J, Murase N, Hara H, Ball S, Loveland BE, Ayares D, Lakkis FG, Cooper DK, Trucco M. Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets. Am J Transplant. 2009;9(12):2716–2726.

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Combined ­pharmacologic therapy with multiple transgenic modifications may prove to be the best method to prevent IBMIR in islet xenotransplantation. Importantly, genetic modifications that delete pig antigens or use promotors to target expression of transgenes in β-cells do not appear to reduce islet function.55,73,160,193,194 The differentiation between IBMIR and rapid ­antibody-mediated rejection of the pig islet graft is unclear and, indeed, they have many similar features. Even if not identical, many of the genetic modifications to reduce IBMIR also reduce antibody-mediated rejection.55,128,195–207 the reduction in antigen expression, and expression of human complement- and ­coagulation-regulatory proteins have all been shown to be protective to pig organs and islets.12,15,20,161–163 If IBMIR and the innate immune response can be controlled by genetic engineering of the islet-source pig, then efforts can be focused on the suppression of the adaptive (cellular) immune response.54,61

Control of the T cell response The genetic manipulations and pharmacologic therapy outlined above attempt to protect against the innate immune system and the early loss of grafts from humoral immunity. However, they do not adequately target prevention of the adaptive immune response (cellular rejection), including the T cell response that causes lymphocytes to infiltrate the graft and induce an elicited antibody response.11,208–210 Moreover, the innate and adaptive immune systems are not mutually exclusive. An organ from a pig genetically modified to combat the innate immune response may also allow reduced immunosuppressive therapy to control the adaptive immune response.73,211–213 For example, there is evidence that the absence of expression of Gal on the graft and the expression of a human complement-regulatory protein both reduce the T cell response.214,215 Nevertheless, it is very likely that some exogenous immunosuppressive therapy will be required to prevent the T cell response to transplanted pig islets and organs for the foreseeable future. T cells require activation through their T cell receptor and peptide-MHC complexes (signal 1) with additional costimulation (signal 2) in order to induce a cellular proliferative response and the secretion of cytokines. Conventional immunosuppressive therapy (directed to blocking the activation and proliferation of resting T cells) has to date proved unsuccessful in fully protecting a pig xenograft.209 In contrast, therapy directed to block T cell costimulation (signal 2), first introduced into pig-to-NHP xenotransplantation by Buhler et al. proved much more

effective.209 Chief among the costimulation-activating pathways is the CD154 (CD40 ligand)-CD40 p ­ athway.216 This provides an attractive target to prevent the adaptive immune response.11,217 Indeed, an anti-CD154 monoclonal antibody (anti-CD154mAb) was the first costimulation blockade agent used in xenotransplantation, and has been employed widely since then in preclinical islet xenotransplantation studies.168,209 Its efficacy is illustrated by the studies of Park and his colleagues who have achieved insulin independence in diabetic monkeys for approximately 2  years by transplanting adult wild-type (WT, i.e., genetically-­ unmodified) pig islets under the cover of anti-CD154mAb therapy (Table  7).24 These remarkable results could not be duplicated when this group replaced anti-CD154mAb therapy with other ­costimulation-blockade agents, including anti-CD40mAb.14,218 In contrast, when genetically engineered whole organ pig grafts are transplanted, anti-CD40mAb-based regimens have proved entirely successful.140,141,219,220 Despite the success of Park’s group using WT pig donors with costimulation blockade-based immunosuppressive therapy, a combination of islet transplantation from multi-transgenic pigs and novel exogenous immunosuppressive therapy holds the most promising future for islet xenotransplantation (Table 7).10,20,24,55,66,193 Together, these can reduce the need for intensive conventional immunosuppressive therapy, ultimately reducing the likelihood of adverse complications, thus reducing the barriers to successful clinical practice.6,80 Although anti-CD154mAb has helped establish normoglycemia from several months to over 2 years in pigto-NHP models, prior Phase I and II clinical trials of organ allotransplantation demonstrated a risk of thromboembolic events, underpinning the balance between efficacy and safety.8,11,12,14,20,112,168,211,213,218,221,222a A recent study, however, demonstrated this agent’s safety in pig-to-NHP islet xenotransplantation, potentially making it a candidate for use in clinical islet transplantation.109 Whether the lack of thromboembolic events after islet xenotransplantation in the pig-to-NHP model was related to the low antigen load of islets (compared with a solid organ) or to other factors remains to be ascertained. Modifications of the molecular structure of this compound aimed at reducing the potential prothrombotic effects are underway and may prove clinically relevant. Anti-CD40mAb is a promising alternative that continues to be explored, particularly when the islets are derived from genetically engineered pigs.11,14,16,168,223–226 Other costimulationblockade agents, for example, CTLA4-Ig, have been found to be less successful when used alone.13,14,227–230 In addition, there are specific genetic modifications that can be made to the pig donor tissues to reduce the

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TABLE 7  Experience with the xenotransplantation of free islets from wild-type pigs in immunosuppressed NHPs genetically-engineered pigs in NHPs (± immunosuppressive therapy) Reference

Donor/recipient

Immunosuppressive therapy

Maximal graft survival (days)

(A) WILD-TYPE PIGS IN IMMUNOSUPPRESSED NHPS Hering et al.8

Adult/CM

FTY720 + rapamycin + anti-IL-2R + anti-CD154

> 187

Cardona et al.11

Neonatal/rhesus monkey

CTLA4-Ig + rapamycin + anti-IL-2R + anti-CD154

> 260

Thompson et al.14

Neonatal/rhesus monkey

CTLA4-Ig + rapamycin + anti-IL-2R + anti-CD40

> 203

16

Neonatal/rhesus monkey

MMF + CTLA4-Ig + LFA-3-Ig + anti-IL-2R + anti-LFA-1

114

Shin et al.

Adult/rhesus monkey

ATG + CVF + rapamycin + anti-TNF + antiCD154mAb(+ Treg)

> 603

Shin et al.218

Adult/rhesus monkey

ATG + CVF + rapamycin + adalimumab + antiCD40mAB(+ tacrolimus or belatacept)

60

Thompson et al. 24

(B) GENETICALLY-ENGINEERED PIGS IN NHPS (± IMMUNOSUPPRESSIVE THERAPY) Mandel et al.163

hCD55 fetal/CM

Cyclosporine + steroids + cyclophosphamide or brequinar > 40

GnT-III adult/CM

None

5

hCD46 adult/CM

MMF + ATG + anti-CD154mAb

> 396

GTKO neonatal/rhesus monkey

MMF + anti-CD154mAb + anti-LFA-1mAb + CTLA4-Ig

249

Chen et al.

GTKO/hCD55/hCD59/hHT neonatal/baboon

MMF + ATG + tacrolimus

28

Bottino et al.20

Multi-transgenic adult/CM

MMF + ATG + anti-CD154mAb

> 365

162

Komoda et al.

12

van der Windt et al. 15

Thompson et al. 161

anti-LFA-1, anti-lymphocyte function-associated antigen-1 monoclonal antibody; ATG, antithymocyte globulin; CM, cynomolgus monkey; GnT-III, Nacetylglucosaminyltransferase-III; hHT, human α(1,2)fucosyltransferase; MMF, mycophenolate mofetil; Treg, autologous regulatory T cell infusion. Modified from (a) Park CG, Bottino R, Hawthorne WJ. Current status of islet xenotransplantation. Int J Surg. 2015;23(Pt B):261–266 and (b) Zhu HT, Yu L, Lyu Y, Wang B. Optimal pig donor selection in islet xenotransplantation: current status and future perspectives. J Zhejiang Univ Sci B. 2014;15(8):681–691.

cellular response, for example, (i) insertion of a mutant (human) MHC class II transactivator gene, resulting in downregulation of swine leukocyte antigen (SLA) class II expression, (ii) deletion of expression of SLA class I (SLA class I-KO), or (iii) insertion of an immunosuppressive gene, for example, CTLA4-Ig55,198,202,230,231 (Table 6).

Will sensitization to human leukocyte antigens be detrimental to islet xenotransplantation? It is well known that patients who have received blood transfusions or organ transplants from human donors, or have been pregnant, can develop antibodies directed toward human leukocyte antigens (HLAs). If the patient then requires an organ or cell transplant, this condition may make it difficult to identify a human donor against which the patient has no preexisting antibodies. There is some evidence that allosensitization to HLAs would not preclude successful xenotransplantation, although there is considerable conflicting evidence in this respect.232,233

Will sensitization to pig antigens preclude subsequent islet allotransplantation? If sensitization develops to a pig xenograft, the limited data available to us at present suggest that the recipient would be at no immunological disadvantage to subsequently undergo allotransplantation when a donor becomes available.232,234,235

The induction of immune tolerance: The “Holy Grail” of transplantation The ultimate goal of islet xenotransplantation is to induce a state in which the host immune system recognizes the transplanted pig islets as “self” and makes no effort to reject them.6 If such immunologic “tolerance” could be achieved, immunosuppressive therapy, which may have detrimental effects on the host (and the graft), could eventually be discontinued.10,61 Xenotransplantation has the advantage that the timing of the transplant is known well in advance, allowing

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manipulation of the host’s immune system (without the constraints of timing that occur in allotransplantation using deceased donors). Nevertheless, we suggest that tolerance induction can only be seriously considered when the early hurdles to pig islet xenotransplantation, for example, IBMIR and the innate immune response, have been fully overcome.80 Most efforts to induce tolerance to both allografts and xenografts have revolved around either (i) donor-specific hematopoietic progenitor cell transplantation in an effort to induce a state of chimerism or (ii) concomitant donor-specific thymus transplantation.236

Improving function of porcine islets Although the pig is considered a good xenogeneic donor, issues remain in establishing sufficient insulin production and glucose control after islet xenotransplantation. The relatively poor secretory function of porcine islets and their relative inability to respond to metabolic stimuli compared to healthy islets from NHPs or humans is often overlooked.73 Pigs utilize less insulin, require lower levels of C-peptide, and maintain higher blood glucose levels compared to NHPs (Table  8).54,160,194,222a Isolated porcine islets secrete three to six times less insulin than human islets when stimulated with glucose in vitro.73,237–240 Unfortunately, this cannot be explained by the lower insulin content of porcine islets, and underscores the field’s poor understanding of porcine β-cell physiology, which has predominately focused on smaller animal models that are more easily obtained.73 Identification of the optimal age and strain to improve proliferation and increase the quantity of islets is only part of the solution.20,73,241 Just as genetic

TABLE 8  Fasting blood glucose, C-peptide, insulin, and glucagon levels in monkeys (Macaca fascicularis), pigs, and humans Cynomolgus monkeys222a

Pigs222a

Humans

Blood glucose (mmol ·L− 1)

2.2–4.1 (3.2)

4.0–5.2 (4.8)

3.9–5.6222b

C-peptide (nmol ·L− 1)

0.47–3.14 (1.39)

0.11–0.32 (0.16)

0.17–0.66222c

Insulin (pmol ·L− 1)

15–201 (109)

7–12 (9)

34–138222c

Glucagon (pmol ·L− 1)

18.7–179.4 (54.3) 11.3–13.8 (12.5)

5.7–28.7222c

Data are presented as ranges (mean). C-peptide (P < .001), insulin (P = .021), and glucagon (P < .001) levels were significantly higher in monkeys than in pigs, while blood glucose levels were significantly (P < .001) lower in monkeys.222a Human data are obtained from the literature and were measured in venous plasma.222b,c Reproduced with permission from Casu A, Bottino R, Balamurugan AN, et al. Metabolic aspects of pig-to-monkey (Macaca fascicularis) islet transplantation: implications for translation into clinical practice. Diabetologia 2008;51(1):120–129.

­ odifications have attempted to reduce IBMIR and islet m graft rejection (described above), so too are they being explored to improve islet function and insulin production. For example, “humanized” pigs have been created that exclusively express human insulin.55,242 Other genetic modifications have attempted to increase insulin secretion by activating insulin granule exocytosis pathways via glucagon-like peptide-1 (GLP1) and cholinergic receptors.55,73,243–245 Pharmacotherapies targeting downstream effectors of these receptors have yet to be explored.73,76,238,246 There is concern that accelerating insulin secretion could in fact exacerbate islet fatigue by accelerating oxygen and nutrient demands in an already metabolically demanding environment.73,77,247 The increased metabolic rates in NHPs and humans could reduce the translational efficacy of preclinical studies.55,224,248 However, if neonatal islets prove to be proliferative in the clinical setting, fewer pigs may be needed, and could potentially combat the problem of islet fatigue. Moreover, if cells fatigue or are injured by lack of oxygen or nutrients, multiple transplants could restore normoglycemia.23,73,193 Because human insulin and blood glucose levels lie between those of pigs and NHPs, a recent consensus statement from the International Xenotransplantation Association (IXA) indicates that successful islet xenotransplantation in NHPs would likely translate to successful clinical trials in humans.54,55,73,224,240,248 Indeed, free islet xenotransplantation has demonstrated normoglycemia in NHPs for approximately 2  years, possibly warranting clinical trials.6,24

Justification for translation to clinical trials Lessons from early clinical trials Continued preclinical investigation using the pigto-NHP model is essential to establish the safety and efficacy of treatment that could translate to clinical success. Despite advancing preclinical studies, to date, with the exception of the study by Groth et  al., free islet xenotransplantation has not been tested in a clinical trial (Table 9).53 After the transplantation of encapsulated pig islets, insulin requirements were modestly reduced in some patients (Table 9).249 However, it is unclear if this was due to successful xenotransplantation, or secondary to meticulous medical management in a controlled research setting (i.e., scrutinized diet, regular glucose monitoring, and routine expert medical attention).249 More recently, Matsumoto et al. improved HbA1c levels to < 7% in patients for > 600  days using encapsulation of porcine islets in the absence of immunosuppressive therapy.23,250 It is important to note the minimal adverse events that

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TABLE 9  Experience with clinical pig islet transplantation (free and encapsulated) Reference

Donor

Immunosuppressive Implantation site therapy

Maximal graft survival Clinical outcome

Groth et al.53

FICCs

Kidney capsule

CsA + prednisolone

Portal vein

CsA + prednisolone + ATG > 460 day + 15-deoxyspergualin

21 day

Plasma porcine C-peptide-negative Urine porcine C-peptide-positive

Valdes-Gonzalez et al.85

NICCs + SCs (encapsulated)

Subcutaneous

None

> 4 yr

Insulin requirement reduced by 50% (in 50%of patients)

Elliott et al.86

FICCs (encapsulated)

Peritoneal cavity

None

> 9 yr

Insulin requirement reduced by 30%

Valdes-Gonzalez et al.88

NICCs + SCs (encapsulated)

Subcutaneous

None

> 3 yr

Insulin requirement reduced from 19-28 IU/d to 6IU/d

Valdes-Gonzalez et al.87

NICCs (encapsulated) Subcutaneous

None

> 7.7 yr

Insulin requirement reduced by 33% (in > 50% of patients)

Wang et al.9

NICCs

CsA + MMF + prednisolone

> 1 yr

Insulin requirement reduced by 33%–62%

Hepatic artery

> 1 yr OKT-3 + tacrolimus  + sirolimus + prednisolone

Insulin requirement reduced 33%–62%

CsA + MMF

Not available

Not available

NICCs (encapsulated) Peritoneal cavity

None

> 52 week

1/14 showed full graft function for a period of time

Matsumoto et al.250 NICCs (encapsulated) Peritoneal cavity

None

> 600 day

HbA1c < 7.0%; reduced insulin and severe episodes of hypoglycemic unawareness

23

Matsumoto et al.

ATG, anti-thymocyte globulin; CsA, cyclosporine; FICCs, fetal islet-like cell clusters; MMF, mycophenolate mofetil; NICCs, neonatal islet-like clusters; SCs, Sertoli cells. MMF = mycophenolate mofetil; NICCs = neonatal islet-like clusters; SCs = Sertoli cells. Modified from Rood PP, Cooper DK. Islet xenotransplantation: are we really ready for clinical trials? Am J Transplant. 2006;6(6):1269–1274.

­ ccurred in these trials, highlighting the safety of clinical o application, but underpinning the need for further studies to improve efficacy.54,251–253

Establishing safety In 2016, the IXA published an executive summary and a seven-chapter consensus statement regarding the prospect of taking porcine islet xenotransplantation into the clinic.54,61,254–259 This was its first update since its original statement in 2009, and highlights new guidelines aimed at accelerating the use of genetically modified pigs. In doing so, their hope was to introduce preclinical guidelines that are less demanding, but maintain patient safety.259 Key to establishing effective clinical trials—indeed a maxim of medicine—is to first ensure safety of the patient and minimize undue risk. Porcine xenotransplants could be associated with zoonotic transmission of microorganisms to the recipient, and possibly even to close contacts and the community. The 2003 US Food and Drug Administration (FDA, updated in 2016) and 2008 First World Health Organization Consultation on Regulatory Requirements for Xenotransplantation

Clinical Trials acknowledged that the possibility of a zoonotic “epidemic” was of sufficient concern when defining the regulatory framework and principal guidelines for xenotransplantation.54,260,261 All pig cell nuclei contain viruses or virus remnants known as porcine endogenous retroviruses (PERV) (just as human cell nuclei contain human endogenous retroviruses [HERV]). These are dormant in their host and not associated with any specific diseases. Despite initial concerns that PERV could become activated in humans, there has never been a report of in vivo infection between pigs and humans.89,262,263 Furthermore, although recent studies have shown successful inactivation of PERV in pig DNA, most of those working in xenotransplantation do not believe this will be necessary.255,264 Today, most experts agree that, using the appropriate precautions, there is minimal risk that porcine xenotransplantation would spread a communicable disease.61,265 Pigs used in clinical trials must be housed in biosecured “designated pathogen-free” facilities that eliminate most potentially pathogenic microorganisms. With good manufacturing practices and established standard operating procedures, the risk is considered minimal.61

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The IXA executive summary outlines recommendations on screening the patient for donor-derived pathogens and pathogens that may be introduced during islet preparation that would ensure safety in well-planned pilot clinical studies. Transplant recipients would not require monitoring for pathogens initially absent from the islet-source pig.61,258,259 The risk of infection secondary to administered immunosuppressive therapy and other risks typical of any transplant or novel therapeutic intervention would require monitoring. These risk-benefit determinations must be acknowledged when obtaining informed consent.56,61,266 In light of their own recommendations, the US FDA will review proposals for clinical trials of xenotransplantation if they contain a plan for patient follow-up.261

Determining efficacy The 2016 IXA guidelines have been slightly revised since 2009 regarding the preclinical justification for transitioning to clinical trials after success in preclinical pigto-NHP models. In 2016, it was suggested that successful treatment without graft rejection be present for at least 6  months, and preferably 12  months, in four of six (or five of eight) consecutive NHPs to warrant clinical trials with potential success in free islet xenotransplantation requiring immunosuppressive therapy.54,61 However, the IXA was hesitant to provide definitive guidelines that might inhibit discussion with regulatory authorities or restrict clinical trials unduly. A minority of experts suggested that a reduced period of graft function (3 months) in NHPs could be sufficient, if the primary goal was improved glycemic control (demonstrated by the absence of recorded hypoglycemic events), rather than a complete insulin independence. However, due to the delayed production of insulin when transplanting embryonic/fetal/ neonatal islets, several months of follow-up might be required to demonstrate reduced insulin requirements or insulin independence.11,13,54 A significant minority of those working in this field also recommended that the requirement for NHP experiments should not be generalized, but rather developed by investigators and regulatory agencies in light of their experience and objectives. Importantly, these shorter durations differ from the US FDA recommendations to conduct animal experiments for 12–24 months.261 Regardless, the IXA acknowledges that preclinical studies using NHPs are at times essential, and always recommended. Similar and perhaps more lenient guidelines were suggested for encapsulated islet xenotransplant studies. Given the difficulty of carrying out studies in NHPs in Europe, the variability in experience and objectives, and the important theoretic ability to transplant encapsulated islets (or islets protected solely by SCs or MSCs) without immunosuppression, a minority of experts

recommended that no work in NHPs may be necessary.54,61 Nevertheless, in either the case of free or encapsulated islet xenotransplantation, preclinical studies should be sufficiently rigorous to establish safety and provide optimism for success in clinical trials, but need not be so demanding as to require prolonged experimentation to ensure success, as this might adversely affect patients who could truly benefit from islet xenotransplantation.54,61,259 It is possible that the lack of preclinical success in NHPs limited the efficacy of past clinical trials.25,53,85,89,267,268 However, it is important to note that the earliest published clinical trials in islet xenotransplantation were performed before the original IXA consensus statement for undertaking clinical trials in 2009.56,61,249,263,269,270 The current guidelines for initiating clinical trials have evolved to keep up with advances in technology, and it is important to recognize that the current era of islet xenotransplantation necessitates established preclinical safety and efficacy in the pig-to-NHP model before proceeding to human trials.61,255,264

Patient selection Defining a clinical study population after preclinical success requires a population with a favorable ­benefit-risk ratio. The US FDA regulations insist that patient selection should focus on, among other criteria, those who “(i) have serious or life-threatening diseases for whom adequately safe and effective alternative therapies are not available except when very high assurance of safety can be demonstrated, and (ii) have potential for a clinically significant improvement with increased quality of life following the procedure.” 61,261,271,272 In light of these guidelines, the IXA recognizes a narrow population of eligible patients that comprises diabetics experiencing recurrent and severe hypoglycemia unawareness despite optimized medical management.61,259 Other potential candidates who have been identified include those with diabetes (with poor glycemic control), end-stage renal disease (who require kidney allotransplantation and might benefit from islet xenotransplantation), and “brittle” diabetics who lack timely access to islet allotransplantation.61,259

Future directions Research priorities The IXA has proposed establishing an IXA Clinical Trial Advisory Committee, whose role would be to advise and serve as an informative body—but not to regulate— for research programs considering initiating clinical trials (and possibly also to advise regulatory agencies on

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References

the scientific aspects of such trials).54 In 2016, IPITA-TTS released a joint executive summary on the future of βcell replacement therapy.6 With regard to islet xenotransplantation, they determined that the first priority is preventing IBMIR with a multifaceted approach that specifically utilizes genetically modified pigs. Second, they encouraged developing an effective and clinically relevant anti-rejection regimen that might be based on disrupting CD40/CD154 signaling.6

Conclusions Roughly 1.25 million Americans have T1Ds, with an additional 40,000 diagnosed each year.273 Progress toward making islet xenotransplantation a clinical reality has grown exponentially. Certainly, hurdles remain, and even success in pilot clinical trials would not guarantee broad applicability. Nevertheless, hope for transplanting tissues across species is no longer quixotic or science fantasy. It is a profound reality that, within the reader’s lifetime, islet xenotransplantation will transform the management of diabetes. In all of this success, we are reminded of Sir Frederick Grant Banting, who shared the Nobel Prize in Medicine and Physiology with John James Rickard Macleod in 1923 for the discovery of insulin. “It is not within the power of the properly constructed human mind to be satisfied. Progress would cease if this were the case.” 274

Acknowledgment Work on xenotransplantation at the University of Alabama at Birmingham is supported in part by NIH NIAID U19 grant AI090959.

Conflict of interest No author declares a conflict of interest.

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